Chromatograph system and method

ABSTRACT

A chromatograph system for a gas-liquid chromatographic analyses of complex samples of matter having a flow regulated carrier gas supply and sample injection means for vaporization and translation through a pre-column and a main analytical column to a detection unit, solvent venting means connected between a precolumn and analytical column for venting a substantial portion of the solvent from the chromatograph during conversion of the sample into detectable form and prior to translation thereof through the analytical column for detection, and means for bypassing a portion of the carrier gas supply into the analytical column downstream of the solvent venting means to facilitate the solvent venting operation. The invention also embodies the method of carrying out analysis of complex samples of matter in gas chromatograph-mass spectrometer combinations utilizing the step of solvent venting subsequent to the sample injection.

United States Patent [1 1 Gehrke et al.

[ May 6,1975

[ CHROM ATOGRAPH SYSTEM AND METHOD [22] Filed: Apr. 28, I972 [21] Appl.No.: 248,384

Primary Examiner.lohn Adee Attorney, Agent, or Firm-Richard G. Heywood[57] ABSTRACT A chromatograph system for a gas-liquid chromatographicanalyses of complex samples of matter having a flow regulated carriergas supply and sample injection means for vaporization and translationthrough a pre-column and a main analytical column to a detection unit,solvent venting means connected between a pre-column and analyticalcolumn for venting a substantial portion of the solvent from thechromatograph during conversion of the sample into detectable form andprior to translation thereof through the analytical column fordetection, and means for by-passing a portion of the carrier gas supplyinto the analytical column downstream of the solvent venting means tofacilitate the solvent venting operation. The invention also embodiesthe method of carrying out analysis of complex samples of matter in gaschromatograph-mass spectrometer combinations utilizing the step ofsolvent venting subsequent to the sample injection.

9 Claims, 2 Drawing Figures CARRIER Q25 10 SUPPLY FLOW CONTROLLERROTOMETER] [52] U.S. Cl... 55/67; 55/]97 [51] ..B01d 15/08 [58] Field ofSearch 55/67, 197

[56] References Cited UNITED STATES PATENTS 3,l 12,028 1/1973 Deans55/l97 X 3,498,027 3/1970 Buchtel, 55/197 3,550,429 12/1970 MacMuataieet al. 551])? X 5 CLOSED cnnmen ans 0.! Aura oven 4/ /O CLOSED OFF AUTOp srnRr PATEHIEUMM sums 3.881.892

sum 20$ 2 DERIVATIZATION AND GLC ANALYSIS OF ng OF EACH AMINO ACID I ORNLYS TYR

(ILA

U LU GL Q VAL Q ASP D Z.

EU a: an L PHE ADJ HyPRO TRY lLE 0 I SER o J L Lu '7 E I I I I I 100 130I60 :50 220 "c ESTERFICATION GLC ANALYSIS SUM-l -Butanob 3NHCL SampleInjected: 50 1 100C-30 Min. Solvent Vent Time: SEC.

Injection Port Temo: 180C ACYLATION Proqram Rate: 6/Min.

Final Temn. 230C 42 /41 CH Cl -IFAA (2 r 1 v/v) Attenuation: 32 x 10 AFS100C-10 Min.

PRE-COLUI N: COLUMN:

1. n w/w% 0V-17 on 8 /1 mesh 0. w/w% EGA on /100 mesh II.P. Chromnscrb G4" x 4 mm. a.w. Chromosorb W, l. 5 m x 4 mm LD.

Glass.

CHROMATOGRAPH SYSTEM AND METHOD BACKGROUND OF THE INVENTION The presentinvention relates to apparatus and methods of analyzing volatilecompounds and more particularly to a sensitive and accurate system foranalysis of separated constituents of mierosized samples.

Analysis of complex samples of matter is carried out by vaporizing aliquid sample in the heated environment of a gas chromatograph. whichseparates the components of the sample into sequential analyticalcomponents and. in a conventional gas chromatograph or like separationapparatus. the gas or vaporized samplc to be analyzed is transportedthrough the chromatograph to the detection unit for analysis by a streamof inert carrier gasv A principal problem in the past has been thatmicrosized sample compounds are diluted in an organic solvent solutionand consequently such small volume samples may be difficult to detect.or the large volume of volatile solvent is immediately swept through theanalytical or separation column and frequently swamps the detector orobscures the sample peaks. Another major obstacle associated with thegasliquid chromatographic analysis of biological samples. or samplescontaining very low concentrations of amino acids or other molecules.has been that the sensitivity ofthe detection means has been minimizedby the limited sample volume that could be injected into a packedanalytical chromatographic column. In many cases. due to problems ofmaintaining intact the derivatives of the biological compounds atextremely low concentrations. problems are encountered. On removal ofthe solvent and excess derivatization reagents. many derivatizedcompounds become susceptible to hydrolysis by atmospheric moisture.Also. on the reduction of the sample volume to a few microliters. thevarious derivatives of the compounds to be analyzed in the solution mayexhibit different solubility characteristics and separate as oils orcrystals. thereby making analyses very difficult or impossible.

OBJECTS AND SUMMARY OF THE INVENTION It is an object ofthe presentinvention to improve the sensitivity and reliability of all kindsofgas-liquid chromatographic analyses. such as steroids. carbohydrates.amino acids. nucleic acids. peptides. drugs. metabolics. volatilemetals. petroleums and the like; and having limited applicability toliquid-liquid and ion-exchange chromatography.

Another object of the invention is the provision of a gas analysissystem capable of accurately analyzing small quantities of sample. butpermitting the introduction of larger volumes of sample into thechromatography column for better sensitivity of detection.

Another object is to provide a chromatographic sys tem that obviates theneed for removal of halogenated solvents from the derivatized sample inelectron cap ture and alkali-flame detection analyses Another object isto provide a chromatographic system that eliminates sul\enl andevtraneous peaks from interferring with early eluted compounds A furtherobject of the invention is the provision of a highly scnsilncchromatgrapliic gas-liquid analysis apparatus capable of flu andtemperature program ming and yet providing accurate and reliablequantitatne data.

These and other objects and advantages of the invention will become moreapparent hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS For purposes ofillustration anddisclosure. the inven tion is embodied in the parts and the combinationof parts hereinafter described and claimed. In the accompanying drawingswhich form a part of the specifica tion:

FIG. I is a diagrammatic view of a gas chromatographic system embodyingthe invention. and

FIG. 2 is a chromatogram resulting from a typical analysis of an aminoacid sample taken in the chromatographic system of FIG. 1.

DESCRIPTION OF THE PREFERRED EMBODIMENT Referring to FIG. I. the presentchromatographic system comprises the usual carrier gas supply I0. whichmaintains an inert gas such as nitrogen. helium or the like underpressure and discharges that gas to a flow controller I] and a rotameterl2 for regulating and reading the rate of gas flow to the system. Thesystem also includes a main chromatograph device l3 having achromatograph column I4 including a pre-column l5 and analytical columnl6 modified according to the present invention. but which chromatographI3 has the conventional controls (not shown] for varying the oventemperature between 50 to 300C or other usual ranges. The analyticalcolumn I6 is connected to a detector unit 17. such as a flame ionizationdetector or a mass spectrometer. for analysis of the eluted compounds.The system also includes an auxiliary chromatograph venting apparatusindicated diagrammab ically at 18. which includes a control panel I).

The carrier gas line from the rotameter 12 connects to a T-connection 2l. and the main gas flow conduit 22 therefrom is connected into thepre-column IS. at 23. adjacent to the sample injection port 24. Theinjection port 24 is conventional in construction and includes aninjector nut 25 sealably connecting a selfsealing rubber disc 26 to theinlet end of the precolumn 15. In practice. the pre-column I5 islengthened so that it can be adapted to fit a number of different basicchromatograph devices 13. Preferably. the chromatographic column I4 ismade of glass (to be inert and responsive to exposed temperatures) and.intcrmediate to the pre-column l5 and analytical column I6, a ventingT-connection 30 is provided. This T- connection 30 is packed with asilanized glass wool plug 3! or the like. and the pro-column l5 andanalytical column I6 are packed in a usual manner with a typi calgranular inert material 32. such as a high molecular weight polymer.above and below the plug 3]. The packing 32 in the pre-column 15 may beI"? O\'-l7 on (hromosorb O marketed by Ohio Valley Specialties and thepacking 32 in the analytical column It: may be 0.65 EGA on (hromosorb Wmarketed by Johns- Manville. which are selected to retainN-trilluoroacetyl n-butyl esters of amino acids v. hile permitting themating ofthc solvent and derivati/ation byproducts. as ill be described,

The 'l'-connection 30 between the pre-column I5 and analytical columnIf) is connected by a tle\ible stainless steel tube conduit 33 or thelike to a solenoid valve 34 in the .iu\iliary unit 18. and an outletconduit 35 from this tube 34 wins to atmosphere. The solenoid valve 34is operated by a timer mechanism 36 set by a manual control 36' on thecontrol panel 19.

An important feature of the present system is the carrier gas hy-passarrangement designed to facilitate the venting operation. A conduit 37is connected to the T- connection 2] in the main carrier gas line 20.22and is connected through a gas metering needle valve 37' in theauxiliary unit 18 to a conduit 38 connected to another T-connection 39downstream of the venting connection 30. both intermediate of thepre-column l5 and analytical column 16. The carrier gas hy-pass lines37.38 are regulated by the needle valve 37' through a control knob 40 onthe control panel I9 to bypass approximately 20% of the carrier gas intothe chromatographic column 14 to assist in the venting operation. aswill he described. The control panel 19 of the auxiliary unit 18 alsoincludes a master on-off" switch 41. a venting switch 42 forconditioning the opening and closing of the timer 36 in conjunction withthe timer control 36. and a timer start button 43 (together withindicator lamps 44 and 45 showing the venting and non-venting conditionsof the auxiliary unit 18).

In operation. the flow controller H is set in the normal manner toregulate the total volumetric flow rate of carrier gas from the carriergas supply it] through the rotameter l2 and conduit 20 to theT-connection 2t. and the gas metering valve 37' in the auxiliary unit 18is adjusted by the control 40 so that approximately It) milliliters perminute of carrier gas (approsimately 20%) pass through the by-pass lines37.38 to the T- connection 39. The timer control 36' is set for thedesired venting time. such as 30 seconds (0.5 as shown in FIG. I l. andappropriate time adjustments may he made depending upon whether there issolvent remaining or the detector peaks of early eluted compounds arelost or their quantities are diminished. Of course. other parameters inthe chromatograph operation that affect solvent and sample separationare column and injection port temperatures. polarity of the columnpacking and the percent loading which are characteristic of gas-liquidchromatographic principles.

The venting switch 42 is turned to open and the sample solution to beanalyzed is then injected into the injection port 24. usually by using ahypodermic sy ringe (not shown) penetrating the disc 26. but any liquidinjection devices may be utilized. Upon injection of the sample. thetimer button 43 is immediately depressed and released to activate thetimer 36 and open the solenoid valve 34. The venting switch 42 is thenturned to auto to control the closing of the solenoid valve 34 at theend of the pre-set venting cycle of the timer control 36'.

L'pon injection of the sample into the heated precolumn 15. the samplesolution is immediately vapori/ed and separates into the constituentcomponents of the sample by reason of their \olatilities and polaritieswhich proceeds slovvly through the pre-column 15 in a normal manner itsa function of the packing and temperaturc. whereas the highly volatilesolvent and estraneous volatile substances from the sample solutionrapidly traverse the pre column 15. With the venting valve 34 in theopen position. these volatile substances and solvent as v\ ell a themain carrier gas tream are di- \erted through the venting conduitfollowing the path of least resistance relative to the packed analyticalcolumn and are vented to atmosphere the bypass carrier gas also flowsthrough the conduits 37.38 into fill the column l4 below the ventingline and. similarly. is diverted upwardly and vented to atmosphere. Thesample compounds of interest proceed slowly through the pre-colurnn t5and are separated from the solvent and other volatile substances. Whenthe timer control 36 times out and the timer 36 closes the solenoidvalve 34 to terminate the venting operation. normal separation occurs asthe carrier gas sweeps the constituent compounds of the sample onthrough the analytical column If: to the detector 17. Thus. the ventingof solvent and volatile reaction byproducts of the sample from thecolumn l4 prevents interference with the normal separation and detectionof the sample compounds of interest. and the column and detector lifeare prolonged.

Referring to FIG. 2. from this chromatograph and the accompanying datasetting out the conditions under which it was obtained. it will bereadily apparent to those skilled in the art of chromatography that theanalysis of such a low concentration as a 10 nanogram solution of aminoacids indicates the high degree of detection sensitivity provided by thepresent invention. It will be understood that in a conventional.unvented gas chromatograph. the detector would be completely swamped bythe solvent and no worthwhile reading could be obtained.

It will be readily apparent to all skilled in the art that theapplicability of the present invention to the principles of gas-liquidchromatography may be equally applicable to certain liquid-liquid orion-exchange chro matography situations and in which essentiallyequivalent rcsults can be obtained.

The invention encompases all changes and modifications of the embodimentdisclosed as will be readily apparent to all skilled in the art. and isonly limited in scope by the claims which follow.

What is claimed is;

I. In combination. a gas chromatographic system having a pre-column witha flow regulated carrier gas supply and injection means through which asample so lution is injected for separation into solvent and samplecompounds and movement thereof through said precolumn. and an analyticalcolumn adapted to receive sample compounds from said pre-column foranalysis in a detection unit; a chromatograph accessory devicecomprising solvent venting means connected interme diate of thepre-column and analytical column. said solvent venting means beingadapted to vent from the precolumn a substantial portion of the carriergas and the solvent from said sample solution during conversion of thesample compounds into a detectable form in the precolumn. said solventventing means including means for controlling said venting operationprior to movement of the sample compounds from the pre-column and pastsaid ttcccssor) dev ice to the analytical column.

2. The chromatographic system according to claim I in which said solventventing means comprises a con duit and valve means for selectivelyclosing said conduit and opening it to atmosphere 3. The chromatographicsystem according to claim 2 in which said last-mentioned means comprisestimer means for controlling the valve means for opening and closing ofsaid Cttlttltltl.

4. The chromatographic sy stem according to claim l in which saidpic-column and analytical column are packed with granularchromatographlc column material. and means for separating the packingmaterial in the respectiv c columns.

5. The chromatograplnc s \\lcm according to claim I including othermeans lot I passing a portion of the carrier gas from said carrier gassupply past said prc column to a point downstream of said sohent ventingmeans.

6. The chromatographic system according to claim 5 in which said solventventing means and said other bypass means are connected between saidpre-column and analytical column in closely adjacent relationship.

7. The chromatographic system according to claim 5 including adjustablevalve means in said hy-pass means for regulating the amount of h \-passcarrier gas.

8. The method of analyzing complex samples of matter in a gaschromatograph having a pre-eolumn and an analytical column. comprisingthe steps of flowing a carrier gas into the pre-column at apredetermined flow rate. injecting a predetermined amount of the samplematter into the pre-column. introducing a minor portion of the carriergas intermediate to the pre-column and analytical column, venting thesolvent and carrier gas to atmosphere from a point intermediate theprecolumn and analytical column for a predetermined time interval, andmoving the vaporized sample constituents through the analytical columnto a detector subsequent to said venting operation.

9. In combination. a chromatographic system comprising first columnmeans having an inlet. a flow regulated carry gas supply for introducinga major portion of carrier gas into the inlet of said first columnmeans. said first column means also having an injection port adapted torecene a sample solution. means in said first column means forseparating said sample solution into its \olatile solvent and componentfractions during movement of said carrier gas and sample solutionthrough said first column means. and second column means sequentiallyarranged with said first column means and being adapted to receive thecomponent fractions of said sample solution from said first columnmeans. and detection means for sensing and analyzing said componentfractions of said sample solution moving through said second columnmeans; a chromatograph accessor) device comprising solvent venting meansincluding an outlet conduit connected intermediate of said first andsecond column means and adapted to vent from said first column means asubstantial amount of said volatile solvent and carrier gas duringseparation and movement of said component fractions of said samplethrough said first column means. timer operated valve means in saidoutlet conduit for controlling the venting operation therethrough. it-pass conduit means connected intermediate of said first and secondcolumn means downstream of said outlet conduit means and upstream ofsaid second column means for hy-passing a minor portion of carrier gaspast said first column means and outlet conduit means. and adjustablevalve means in said hy-pass conduit means for regulating the amount ofhy-pass carrier gas therethrough.

1. IN COMBINATION, A GAS CHROMATOGRAPHIC SYSTEM HAVING A PER-COLUMN WITHA FLOW REGULATED CARRIER GAS SUPPLY AND INJECTION MEANS THROUGH WHICH ASAMPLE SOLUTION IS INJECTED FOR SEPARATION INTO SOLVENT AND SAMPLECOMPOUNDS AND MOVEMENT THEREOF THROUGH SAID PRE-COLUMN, AND ANANALYTICAL COLUMN ADAPTED TO RECEIVE SAMPLE COMPOUNDS FROM SAIDPRE-COLUMN FOR ANALYSIS IN A DETECTION UNIT; A CHROMATOGRAPH ACCESSORYDEVICE COMPRISING SOLVENT VENTING MEANS CONNECTED INTERMEDIATE OF THEPRE-COLUMN AND ANALYTICAL COLUMN, SAID SOLVENT VENTING MEANS BEINGADAPTED TO VENT FROM THE PRE-COLUMN A SUBSTANTIAL PORTION OF THE CARRIERGAS AND THE SOLVENT FROM SAID SAMPLE SOLUTION DURING CONVERSION OF THESAMPLE COMPOUNDS INTO A DETECTABLE FORM IN THE PRECOLUMN, SAID SOLVENTVENTING MEANS INCLUDING MEANS FOR CONTROLLING SAID VENTING OPERATIONPRIOR TO MOVEMENT OF THE SAMPLE COMPOUNDS FROM THE PRECOLUMN AND PASTSAID ACCESSORY DEVICE TO THE ANALYTICAL COLUMN.
 2. The chromatographicsystem according to claim 1 in which said solvent venting meanscomprises a conduit and valve means for selectively closing said conduitand opening it to atmosphere.
 3. The chromatographic system according toclaim 2 in which said last-mentioned means comprises timer means forcontrolling the valve means for opening and closing of said conduit. 4.The chromatographic system according to claim 1 in which said pre-columnand analytical column are packed with granular chromatographic columnmaterial, and means for separating the packing material in therespective columns.
 5. The chromatographic system according to claim 1including other means for by-passing a portion of the carrier gas fromsaid carrier gas supply past said pre-column to a point downstream ofsaid solvent venting means.
 6. The chromatographic system according toclaim 5 in which said solvent venting means and said other by-pass meansare connected between said pre-column and analytical column in closelyadjacent relationship.
 7. The chromatographic system according to claim5 including adjustable valve means in said by-pass means for regulatingthe amount of by-pass carrier gas.
 8. The method of analyzing complexsamples of matter in a gas chromatograph having a pre-column and ananalytical column, comprising the steps of flowing a carrier gas intothe pre-column at a predetermined flow rate, injecting a predeterminedamount of the sample matter into the pre-column, introducing a minorpoRtion of the carrier gas intermediate to the pre-column and analyticalcolumn, venting the solvent and carrier gas to atmosphere from a pointintermediate the pre-column and analytical column for a predeterminedtime interval, and moving the vaporized sample constituents through theanalytical column to a detector subsequent to said venting operation. 9.In combination, a chromatographic system comprising first column meanshaving an inlet, a flow regulated carry gas supply for introducing amajor portion of carrier gas into the inlet of said first column means,said first column means also having an injection port adapted to receivea sample solution, means in said first column means for separating saidsample solution into its volatile solvent and component fractions duringmovement of said carrier gas and sample solution through said firstcolumn means, and second column means sequentially arranged with saidfirst column means and being adapted to receive the component fractionsof said sample solution from said first column means, and detectionmeans for sensing and analyzing said component fractions of said samplesolution moving through said second column means; a chromatographaccessory device comprising solvent venting means including an outletconduit connected intermediate of said first and second column means andadapted to vent from said first column means a substantial amount ofsaid volatile solvent and carrier gas during separation and movement ofsaid component fractions of said sample through said first column means,timer operated valve means in said outlet conduit for controlling theventing operation therethrough, by-pass conduit means connectedintermediate of said first and second column means downstream of saidoutlet conduit means and upstream of said second column means forby-passing a minor portion of carrier gas past said first column meansand outlet conduit means, and adjustable valve means in said by-passconduit means for regulating the amount of by-pass carrier gastherethrough.